A novel quantification strategy of transferrin and albumin in human serum by species-unspecific isotope dilution laser ablation inductively coupled plasma mass spectrometry (ICP-MS)

Species-specific (SS) isotope dilution analysis with gel electrophoresis (GE)-laser ablation (LA)-ICP-MS is a promising technique for the quantification of particular metal-binding proteins in biological samples. However, unavailable isotopically enriched spike and metal losses in GE separation are main limitations for SS-isotope dilution PAGE-LA-ICP-MS. In this study, we report for the first time the absolute quantification of transferrin (Tf) and albumin (Alb) in human serum by non-denaturing (native) GE combined with species-unspecific isotope dilution mass spectrometry (IDMS). In order to achieve a homogeneous distribution of both protein and isotope-enriched spike (simulated isotope equilibration), immersing the protein strips with ³⁴S spike solution after gel electrophoresis was demonstrated to be an effective way of spike addition. Furthermore, effects of immersion time and ³⁴S spike concentration were investigated to obtain optimal conditions of the post-electrophoresis isotope dilution method. The relative mass of spike and ablated sample (m_sp/m_sam) in IDMS equation was calculated by standard Tf and Alb proteins, which could be applied to the quantification of Tf and Alb in ERM-DA470k/IFCC for method confirmation. The results were in agreement with the certified value with good precision and small uncertainty (1.5–3%). In this method, species-specific spike protein is not necessary and the integrity of the heteroatom-protein could be maintained in sample preparation process. Moreover, the application of species-unspecific isotope dilution GE-LA-ICP-MS has the potential to offer reliable, direct and simultaneous quantification of proteins after conventional 1D and 2D gel electrophoretic separations.     

Lead speciation in rainwater by isotope dilution-high performance liquid chromatography-inductively coupled plasma-mass spectrometry

A new method for lead speciation in rainwater by isotope dilution analysis (IDA) using directly coupled high performance liquid chromatography-inductively coupled plasma-mass spectrometry (HPLC-ICP-MS) is described and demonstrated. Samples containing trimethyllead (TML) chloride and triethyllead (TEL) chloride in the presence of large amounts of inorganic lead have been analysed by HPLC-ICP-MS using reverse phase ion-pairing chromatography. The detection limit for the procedure is 3 ng g⁻¹ for TML as Pb and 14 ng g⁻¹ for TEL as Pb, with a linear range exceeding 1000 ng g⁻¹ and a relative standard deviation of 4% for TML in the range 50–1000 ng g⁻¹. TML chloride isotopically enriched in the stable 206 isotope was prepared and used to enhance the accuracy of the method by isotope dilution analysis. The IDA-HPLC-ICP-MS method was successfully applied to the analysis of a sample of artificial rainwater used in a collaborative study.     

Absolute quantification of superoxide dismutase (SOD) using species-specific isotope dilution analysis

Here we report for the first time the use of species-specific isotope dilution mass spectrometry for the absolute quantification of a metalloprotein using non-denaturing gel electrophoresis laser ablation inductively coupled plasma mass spectrometry (GE-LA-ICP-MS). The concept utilises the intrinsic metals of the metalloprotein for labelling of the isotopically labelled spike (⁶⁵Cu, ⁶⁸Zn SOD). The stability of the metal–protein complex under non-denaturing conditions during 1-D PAGE was confirmed and the performance of the method evaluated. Between 4 and 64 μg, SOD was quantified with a recovery rate between 82% and 110% in a standard. The use of the isotopically enriched SOD was utilised to identify the extent of orthogonal diffusion in 1-D gel electrophoresis. Orthogonal diffusion of natural and isotopically enriched SOD in the gel can interfere with the correct determination of the isotope ratios. The matrix effect of a cytosolic liver extract on the non-covalently bound copper and zinc in SOD was evaluated and no significant metal loss from the SOD spike was observed. This study represents the first step necessary for establishing and evaluating the use of a species-specific isotope dilution approach for the absolute quantification of SOD in real samples based on the combination of gel electrophoresis and LA-ICP-MS.     

Isotope dilution quantification of ultratrace gamma-glutamyl-Se-methylselenocysteine species using HPLC with enhanced ICP–MS detection by ultrasonic nebulisation or carbon-loaded plasma

A method for the accurate determination of ultratrace selenium species of relevance to cancer research, such as gamma-glutamyl-Se-methylselenocysteine (γ-glutamyl-SeMC), using species-specific double isotope dilution analysis (IDA) with HPLC–ICP–MS is reported for the first time. The ⁷⁷Se-enriched γ-glutamyl-SeMC spike was produced in-house by collecting the fraction at the retention time of the γ-glutamyl-SeMC peak from a chromatographed aqueous extract of ⁷⁷Se-enriched yeast, pooling the collected fractions and freeze-drying the homogenate. The Se content of this spike was characterised using reverse isotope dilution mass spectrometry (IDMS) and the isotopic composition of this spike was checked prior to quantification of the natural abundance dipeptide species in garlic using speciated IDA. The extraction of the γ-glutamyl-SeMC species in water was performed in a sonication bath for 2 h after adding an appropriate quantity of ⁷⁷Se-enriched γ-glutamyl-SeMC to 50 mg of garlic to give optimal ⁷⁸Se/⁷⁷Se and ⁸²Se/⁷⁷Se ratios of 1.5 and 0.6, respectively. The effect of ultrasonic nebulisation, in comparison with the loading of the ICP with carbon (through the addition of methane gas on-line), on the detection of Se associated with γ-glutamyl-SeMC using collision/reaction cell ICP–MS with hydrogen as collision gas was investigated. Sensitivity enhancements of approximately fourfold and twofold were achieved using USN and methane mixed plasma, respectively, in comparison with conventional nebulisation and conventional Ar ICP–MS. However, an approximately twofold improvement in the detection limit was achieved using both approaches (42 ng kg⁻¹ for ⁷⁸Se using peak height measurements). The use of species-specific IDMS enabled quantification of the dipeptide species at ng g⁻¹ levels (603 ng g⁻¹ Se) in the complex food matrix with a relative standard deviation (RSD, n = 4) of 4.5%, which was approximately half that obtained using standard addition as a confirmatory technique. Furthermore, good agreement was found between the γ-glutamyl-SeMC species concentrations obtained using both calibration methods.     

Old commercialized magnetic particles new trick: Intrinsic internal standard

Magnetic particles（MPs） are the most widely used commercialized engineering particles,which gained great success in various biological applications.Inspired by their intrinsic Fe isotope composition,we discovered a commercialized MPs-internal standard’s novel function to realize the accurate quantification of biomolecules.The bioassay of carcinoembryonic antigen（CEA） was chosen as a modal system.The Fe isotope in MPs and Au isotope in report probes were simultaneously and sensitively detected by...     

Development of cadmium/silver/palladium separation by ion chromatography with quadrupole inductively coupled plasma mass spectrometry detection for off-line cadmium isotopic measurements

A separation method was investigated to perform off-line cadmium isotopic measurements on a ¹⁰⁹Ag transmutation target. Ion chromatography (IC) with Q ICPMS detection (quadrupole inductively coupled plasma mass spectrometry detection) was chosen to separate cadmium from the isobarically interfering elements, silver and palladium, present in the sample. The optimization of chromatographic conditions was particularly studied. Several anion and cation columns (Dionex AG11^®, CS10^® and CS12^®) were compared with different mobile phases (HNO₃, HCl). The separation procedure was achieved with a carboxylate-functionalized cation exchange CS12 column using 0.5 M HNO₃ as eluent. The developed technique yielded satisfactory results in terms of separation factors (greater than 5) and provides an efficient solution to obtain rapidly purified cadmium fractions (decontamination factors higher 100,000 for silver and palladium) which can directly be analyzed by multi collection inductively coupled plasma mass spectrometry (MC ICPMS). By applying the proposed procedure, accurate and precise cadmium isotope ratios were determined for the irradiated ¹⁰⁹Ag transmutation target.     

SEC-ICP-MS and on-line isotope dilution analysis for characterisation and quantification of immunochemical assays

This study presents a novel size exclusion chromatography-inductively coupled plasma-mass spectrometry (SEC-ICP-MS) method for the characterisation and quantification of immunoassays with lanthanide-labelled antibodies. SEC-ICP-MS in combination with a double isotope dilution approach enabled facile validation of the antibodies’ integrity, the determination of the batch to batch labelling efficiency, monitoring of each labelling step, and quantification of the immunocomplexes after incubation with the target protein. The addition of oxygen into the dynamic reaction cell improved the detection of sulphur as a marker for the antibodies and target protein via mass-shifting (LOD = 3.7 ng/mL), whilst maintaining sufficient sensitivity for the analysis of the lanthanides. Ultra-high performance liquid chromatography (UHPLC) SEC ensured a rapid chromatographic method with separation times under 7 min of the labelled and unlabelled antibodies, the immunocomplexes, and the unconjugated polymer used to lanthanide-label the antibodies. SEC calibration estimated the molecular weights of all peaks and provided valuable insights in immunochemical reactions and the stoichiometry of the reactants and products. A novel on-line isotope dilution analysis (IDA) enabled absolute quantification of sulphur and lanthanide signals and the protein of interest. The chromatographic separation of immunocomplexes and labelled antibodies allowed the simultaneous determination of the antibody/metal stoichiometry and target protein concentration from a single mass flow chromatogram. An immunoglobulin protein was quantified after incubation with an ¹⁵³Eu-labelled primary polyclonal antibody. The procedure was validated with direct labelling of the target protein with ¹⁵⁶Gd for parallel, simultaneous quantification. The concentration determined via direct labelling of the protein deviated 1.9% from the immunochemical approach employing ¹⁵³Eu-labelled polyclonal antibodies.

Graphical abstract

     

Quantitative TLC Analysis of Sterol (24β-Ethylcholesta-5,22E,25-triene-3β-ol) in Agnimantha (Clerodendrum phlomidis Linn)

A quantitative method using silica gel 60F₂₅₄ high performance thin layer chromatography plates, automated bandwise sample application, and automated visible mode densitometric method has been developed for the determination of 24β-ethylcholesta-5,22E,25-triene-3β-ol (ECTO) in the aerial part of Clerodendrum phlomidis. ECTO was used as a chemical marker for the standardization of C. phlomidis plant extracts. The separation was performed on silica gel 60F₂₅₄ TLC plates using chloroform-methanol (98.5: 1.5, v/v) as mobile phase. The quantitation of ECTO was carried out using the densitometric reflection/absorption mode at 650 nm after post chromatographic derivatization with anisaldehyde reagent. A precise and accurate quantification can be performed in the linear working concentration range of 150–400 ng band⁻¹ with good correlation (r ² = 0.996). The method was validated for peak purities, precision, robustness, limit of detection (LOD) and quantitation (LOQ), etc. as per ICH guidelines.     

Parallel detection,quantification, and depth profiling of peptides with dynamic-secondary ion mass spectrometry (D-SIMS) ionized by C60–Ar co-sputtering

Time-of-flight secondary ion mass spectrometry (ToF-SIMS) using pulsed C₆₀⁺ primary ions is a promising technique for analyzing biological specimens with high surface sensitivities. With molecular secondary ions of high masses, multiple molecules can be identified simultaneously without prior separation or isotope labeling. Previous reports using the C₆₀⁺ primary ion have been based on static-SIMS, which makes depth profiling complicated. Therefore, a dynamic-SIMS technique is reported here. Mixed peptides in the cryoprotectant trehalose were used as a model for evaluating the parameters that lead to the parallel detection and quantification of biomaterials. Trehalose was mixed separately with different concentrations of peptides. The peptide secondary ion intensities (normalized with respect to those of trehalose) were directly proportional to their concentration in the matrix (0.01–2.5 mol%). Quantification curves for each peptide were generated by plotting the percentage of peptides in trehalose versus the normalized SIMS intensities. Using these curves, the parallel detection, identification, and quantification of multiple peptides was achieved. Low energy Ar⁺ was used to co-sputter and ionize the peptide-doped trehalose sample to suppress the carbon deposition associated with C₆₀⁺ bombardment, which suppressed the ion intensities during the depth profiling. This co-sputtering technique yielded steadier molecular ion intensities than when using a single C₆₀⁺ beam. In other words, co-sputtering is suitable for the depth profiling of thick specimens. In addition, the smoother surface generated by co-sputtering yielded greater depth resolution than C₆₀⁺ sputtering. Furthermore, because C₆₀⁺ is responsible for generating the molecular ions, the dosage of the auxiliary Ar⁺ does not significantly affect the quantification curves.     

Evaluation of Manganese(II) and Manganese(VII) Speciation in Water Samples by Ion Pair High-performance Liquid Chromatography-inductively Coupled Plasma Mass Spectrometry

The speciation of Mn(II) and Mn(VII) is reported by ion pair chromatography. To optimize the separation, sample pH, ion pair reagent, Mn(II) complexing agent, and composition of mobile phase were characterized. The separation of Mn(II) and Mn(VII) was performed using ethylenediamine tetraacetic acid to complex Mn(II), tetrabutylammonium hydroxide as an ion pair reagent, and a C8 column. The separation of the manganese species was demonstrated by high-performance liquid chromatography-inductively coupled plasma mass spectrometry (HPLC ICP-MS). The conversion of Mn(VII) to Mn(II) occurred during the separation and influenced the quantification; hence, the factors affecting this process including the storage time, manganese species ratio, and sample matrix composition were evaluated and suitable calibration was demonstrated. The method was validated by characterization of the selectivity, specificity, linearity, limits of detection and quantification, repeatability, and intermediate precision. The detection limit for Mn(II) was 0.22 µg?L^?1, while for Mn(VII), the value was 1.55 µg?L^?1.     

Global quantification of phosphoproteins combining metabolic labeling and gel‐based proteomics in B. pumilus

Differential proteomics targeting the protein abundance is commonly used to follow changes in biological systems. Differences in localization and degree of post‐translational modifications of proteins including phosphorylations are of tremendous interest due to the anticipated role in molecular regulatory processes. Because of their particular low abundance in prokaryotes, identification and quantification of protein phosphorylation is traditionally performed by either comparison of spot intensities on two‐dimensional gels after differential phosphoprotein staining or gel‐free by stable isotope labeling, sequential phosphopeptide enrichment and following LC‐MS analysis. In the current work, we combined in a proof‐of‐principle experiment these techniques using ¹⁴N/¹⁵N metabolic labeling with succeeding protein separation on 2D gels. The visualization of phosphorylations on protein level by differential staining was followed by protein identification and determination of phosphorylation sites and quantification by LC‐MS/MS. This approach should avoid disadvantages of traditional workflows, in particular the limited capability of peptide‐based gel‐free methods to quantify isoforms of proteins. Comparing control and stress conditions allowed for relative quantification in protein phosphorylation in Bacillus pumilus exposed to hydrogen peroxide. Altogether, we quantified with this method 19 putatively phosphorylated proteins.     

Determination of new pyridoquinoline derivatives and their quantification in urine by capillary liquid chromatography

Capillary liquid chromatography (CLC) in reversed separation mode was applied to the quantification and impurity profile determination of eight newly synthesized pyridoquinolines. The CLC separation system consisted of Nucleosil C18 stationary phase and methanol containing 1% (v/v) of triethylamine (TEA) as the mobile phase. The optimized separation system enabled the separation of impurities from the main component and their quantification in a reasonable analysis time. The presence of TEA in the mobile phase greatly improved the peak shape and decreased the analysis time of the studied derivatives on the C18 stationary phase. Calibration curves of pyridoquinolines were plotted in a concentration range from 1.0×10^–6 to 1.0×10^–3 mol/L at two parallel detector wavelengths of 254 and 265 nm and subsequently used for quantification of pyridoquinoline derivatives in human urine. No pretreatment of the real biological sample was needed. Detection and quantification limits were calculated for all the derivatives and the detection limits of most of the pyridoquinolines were found to lie in the μmol/L concentration range. The proposed CLC separation system has proved to be a suitable method for quantification of test derivatives in samples containing human urine matrix.     

Selective Detection of Peptide-Oligonucleotide Heteroconjugates Utilizing Capillary HPLC-ICPMS

A method for the selective detection and quantification of peptide:oligonucleotide heteroconjugates, such as those generated by protein:nucleic acid cross-links, using capillary reversed-phase high performance liquid chromatography (cap-RPHPLC) coupled with inductively coupled plasma mass spectrometry detection (ICPMS) is described. The selective detection of phosphorus as ³¹P⁺, the only natural isotope, in peptide-oligonucleotide heteroconjugates is enabled by the elemental detection capabilities of the ICPMS. Mobile phase conditions that allow separation of heteroconjugates while maintaining ICPMS compatibility were investigated. We found that trifluoroacetic acid (TFA) mobile phases, used in conventional peptide separations, and hexafluoroisopropanol/triethylamine (HFIP/TEA) mobile phases, used in conventional oligonucleotide separations, both are compatible with ICPMS and enable heteroconjugate separation. The TFA-based separations yielded limits of detection (LOD) of ~40 ppb phosphorus, which is nearly seven times lower than the LOD for HFIP/TEA-based separations. Using the TFA mobile phase, 1–2 pmol of a model heteroconjugate were routinely separated and detected by this optimized capLC-ICPMS method.     

Super-Paramagnetic Nanoparticles by Surface Imprinting on Graphene Oxide Modified Iron (II,III) with Application for the Determination of Ovalbumin by Absorption Spectroscopy

Protein surface imprinting produces materials capable of selective recognition and capture of proteins. Herein, a protein surface imprinted polymer on graphene oxide modified super-paramagnetic Fe₃O₄ nanoparticles is reported. The molecularly imprinted polymer was synthesized by ultrasound-assisted suspension polymerization, using ovalbumin as the template molecule, 3-aminophenylboronie acid as the functional monomer, and methylene-bis-acrylamide as the cross-linking agent. The nanoparticles were approximately 40 nanometers in size and super-paramagnetic. Moreover, these particles demonstrated considerably high adsorption capacity, fast adsorption kinetics, and selective binding affinities toward the template protein ovalbumin. The calibration curve of ovalbumin was linear from 5.0 × 10^?11 to 1.0 × 10^?10 molar. The limit of detection of ovalbumin was 2.0 × 10^?11 M. These results show that this super-paramagnetic material has potential for biological macromolecule separation and determination.     

Measurement of uranium isotope ratios in solid samples using laser ablation and diode laser-atomic absorption spectrometry

A diode laser was used for the selective detection of ²³⁵U and ²³⁸U in a laser-induced plasma ignited by a Nd:YAG laser beam focused onto uranium oxide samples. The diode laser was sequentially tuned to the absorption lines of both isotopes (682.6736 nm for ²³⁵U, and 682.6913 nm for ²³⁸U). The absorption was measured on a pulse-to-pulse basis; the transient absorption peak was used as an analytical signal. Three samples were used with the relative abundance of the minor isotope ²³⁵U of 0.204%, 0.407% and 0.714%. Optimal conditions for the detection of the minor isotope were obtained at a distance of ∼3 mm from the sample surface, an argon pressure of ∼3 kPa and for 7.5 mJ pulse energy of the Nd:YAG laser. Absorption in the wing of the broadened line of the ²³⁸U isotope was found to be the main source of background for the measurement of the absorption of the minor isotope. The limit of detection of the minor isotope, evaluated on the basis of the 3σ criteria was estimated to be 100 μg g⁻¹. At the optimal conditions for the detection of the minor isotope optical thick conditions in the line centre of the main isotope were observed. Therefore, the isotope ratio measurements were performed by rationing the intensity of the net absorption signal measured in the line centre of the minor isotope and the absorption signal measured in the wing of the main isotope. This strategy was checked by determination of the isotope ratios for the two samples with depleted ²³⁵U concentration using the sample with the natural isotope composition (0.714%) as a standard. The accuracy and precision for this measurement strategy was evaluated to approximately 10%.     

Ion chromatography inductively coupled plasma mass spectrometry (IC-ICP-MS) and radiometric techniques for the determination of actinides in aqueous leachate solutions from uranium oxide

The choice of the analytical method for the determination of actinide isotopes in leachate solutions has to be made considering several parameters: detection limit for each isotope, sample preparation procedure in terms of duration and complexity, counting time and interferences. A leachate solution obtained by keeping a pellet of UO₂ doped with ²³⁸Pu in contact with distilled water was investigated for the content of U and Pu isotopes by radiometric methods (α-, γ-spectrometry and liquid scintillation counting). The results of the radiometric methods were compared with those obtained from the analysis performed by inductively coupled plasma mass spectrometry on-line to a system for chromatographic separation (IC-ICP-MS). The comparison confirmed that IC-ICP-MS is a powerful method for the detection of long-lived radionuclides. The radiometric methods have a detection limit two orders of magnitude lower than IC-ICP-MS in the case of short-lived radioisotopes mostly due to the low background in the detector. On the other hand, the sample preparation and the analysis duration are more time-consuming compared to IC-ICP-MS; moreover, not all isotopes can be determined by using only one radiometric technique.     

Preparation of core–shell structure Fe3O4@SiO2 superparamagnetic microspheres immoblized with iminodiacetic acid as immobilized metal ion affinity adsorbents for His‐tag protein purification

The core–shell structure Fe₃O₄/SiO₂ magnetic microspheres were prepared by a sol–gel method, and immobiled with iminodiacetic acid (IDA) as metal ion affinity ligands for protein adsorption. The size, morphology, magnetic properties and surface modification of magnetic silica nanospheres were characterized by various modern analytical instruments. It was shown that the magnetic silica nanospheres exhibited superparamagnetism with saturation magnetization values of up to 58.1 emu/g. Three divalent metal ions, Cu²⁺, Ni²⁺ and Zn²⁺, were chelated on the Fe₃O₄@SiO₂–IDA magnetic microspheres to adsorb lysozyme. The results indicated that Ni²⁺‐chelating magnetic microspheres had the maximum adsorption capacity for lysozyme of 51.0 mg/g, adsorption equilibrium could be achieved within 60 min and the adsorbed protein could be easily eluted. Furthermore, the synthesized Fe₃O₄@SiO₂–IDA–Ni²⁺ magnetic microspheres were successfully applied for selective enrichment lysozyme from egg white and His‐tag recombinant Homer 1a from the inclusion extraction expressed in Escherichia coli. The result indicated that the magnetic microspheres showed unique characteristics of high selective separation behavior of protein mixture, low nonspecific adsorption, and easy handling. This demonstrates that the magnetic silica microspheres can be used efficiently in protein separation or purification and show great potential in the pretreatment of the biological sample. Copyright © 2015 John Wiley & Sons, Ltd.     

HPLC for Separation and Quantification of Deoxyribonucleic Acid Fragments and Measurement of Deoxyribonucleic Acid Degradation

DNA quantification has made its mark in pharmaceutical analysis and the life sciences. In particular, in the quality control of nucleic acid drugs and the detection and quantification of genetically modified organisms, evaluation of the DNA degradation rate has become imperative. In this study, by using high-performance liquid chromatography with an anion-exchange column, we established a method for the separation and quantification of DNA fragments in mixed DNA samples. By using a NaCl concentration gradient, DNA fragments in mixed DNA sample were separated well. A calibration curve from 0.05 to 12.4 ng μL^?1 was obtained with high linearity and the correlation coefficient was 0.9999. The limit of detection was 0.02 ng μL^?1 and the limit of quantification was 0.06 ng μL^?1 for S/N = 3 or S/N = 10, respectively. The relative standard deviation was less than 2 % in the measurement of peak area repeatability. The recovery of approximately 1 ng μL^?1 of a specific DNA spiked in a mixed DNA sample was 99.9 ± 3.6 %. The method was able to measure the degradation rate of 600 bp DNA with a variation of approximately 1 %.     

The role of sulfur and sulfur isotope dilution analysis in quantitative protein analysis

The element sulfur is almost omnipresent in all natural proteomes and plays a key role in protein quantification. Incorporated in the amino acids cysteine and methionine, it has been served as target for many protein-labeling reactions in classic quantitative proteomic approaches based on electrospray or MALDI mass spectrometry. This critical review discusses the potential and limitations of sulfur isotope dilution analysis (IDA) by inductively coupled plasma—mass spectrometry (ICP-MS) for absolute protein quantification. The development of this approach was made possible due to the improved sensitivity and accuracy of sulfur isotope ratio measurement by ICP-MS in recent years. The unique feature of ICP-MS, compound-independent ionization, enables compound (species)-unspecific sulfur IDA. This has the main advantage that only one generic sulfur standard (i.e., one isotopically labeled sulfur spike) is required to quantify each peptide or protein in a sample provided that they are completely separated in chromatography or electrophoresis and that their identities are known. The principles of this approach are illustrated with selected examples from the literature. The discussion includes also related fields of P/S and metal/S ratio measurements for the determination of phosphorylation degrees of proteins and stoichiometries in metalloproteins, respectively. Emerging new areas and future trends such as protein derivatization with metal tags for improved sensitivity of protein detection in ICP-MS are discussed. Figure The key role of sulfur in protein quantification     

Development of a CE‐MS2 method for the enantiomeric separation of L/D‐carnitine: Application to the analysis of infant formulas

A new chiral analytical method based on CE‐MS is proposed for the identification and simultaneous quantification of D /L ‐carnitine in infant formulas. Previous derivatization of carnitine with FMOC enabled the optimization of the chiral separation using CE with UV detection. An optimization of electrospray‐MS parameters using a partial filling of the non‐volatile chiral selector (succinyl‐γ‐CD) was performed. A selective fragmentation using MS² experiments with an ion trap analyser was carried out to confirm the identity of D /L ‐carnitine according to the current legislation. Satisfactory results were obtained in terms of linearity, precision, and accuracy. Interestingly, the CE‐MS² method developed allowed a sensitivity enhancement with respect to UV detection of 100‐fold, obtaining an LOD of 100 ng/g for D ‐carnitine. The determination of L ‐carnitine and its enantiomeric purity in 14 infant formulas supplemented with carnitine was successfully achieved, sample preparation only requiring an ultrafiltration with centrifugal filter devices to retain the components with the highest molecular weights.